Risk of allergy and nutrition to reduce that risk

ABSTRACT

A method to predict the risk of eczema is provided based on differences in the development of microbiota and its metabolites in healthy infants and infants that develop eczema, and nutritional remedies based on this finding, in the form of lactate utilizing bacteria and fibres stimulating lactate utilizing bacteria.

FIELD OF THE INVENTION

The present invention is in the field of allergy, in particular eczema,and concerns a method to assess early in life the risk of developingallergy. Also the invention concerns nutritional remedies to reduce therisk of allergy.

BACKGROUND OF THE INVENTION

Early gut microbial development is a dynamic process that may influencehuman health throughout life. Aberrations in this process have beenassociated with the development of allergic diseases, but exactmicrobial patterns remain unclear. The gradual diversification towards arelatively stable adult-like composition is a dynamic process influencedby environmental factors such as birth mode, gestational age at birthand early life nutrition. The importance of pioneering microbes isparticularly clear as alterations in the colonization process have beenassociated with the rise of allergic disorders in affluent societies.Studies so far point into the direction that the gut microbiota ofallergic infants exhibit an accelerated ecological succession to acommunity structure more typical of adults. Allergic diseases in theirdifferent manifestations comprise the most common chronic disease inchildhood in developed countries. Atopic dermatitis is typically thefirst allergic manifestation with a sequential onset to other allergicmanifestations, a process known as the allergic march, also called theatopic march.

First facultative anaerobic bacteria that grow fast on simple sugars,such as Enterobacteriaceae, are gradually replaced by anaerobicbacteria, such as the genera Bifidobacterium and Bacteroides. Themicrobiota further diversifies with the introduction of solid foods tothe infant diet at 4-6 months of age, which establishes more strictanaerobes, such as the adult-type Clostridium clusters IV and XIVa. Thesuccession of species and establishment of a stable adult-type communityin early life is guided by human milk and may be critically important,both from microbial composition and activity point of view, as in viewof the concurrent maturation of the immune system. This earlycolonization and succession is thought to form a crucial process layingthe foundation for optimal health later in life.

Breastfeeding is thought to protect against the development of allergy,as numerous allergens and immune mediators present in human milk areabsent from artificial milks, as well as the high abundance of humanmilk oligosaccharides. Several studies have been performed withdifferent types of infant formulas (IF) supplemented with non-digestibleoligosaccharides, known as prebiotics. Prebiotics are typicallynon-digestible fibres that reach the colon intact and are known toselectively stimulate the growth and activity of specific beneficialmembers of the microbiota. Significant reductions in eczema risk wereobserved in intervention trials with IF supplemented with specificprebiotic oligosaccharides (Arslanoglu et al., 2012, J Biol Regulation &Homeostatic Agents 26:49-59; Grüber et al., 2010, J Allergy and ClinicalImmunology 126:791-797; Moro et al., 2006, Archives of Disease inChildhood 91:814-819.

WO 2009/102199 discloses compositions comprising bifidobacteria andgalacto-oligosaccharides and fructo-oligosaccharides that support theintroduction of solid weaning foods in the diet of infants that receiveinfant milk formula or breast milk. US 2015/0352162 disclosescompositions, particularly baby food compositions, comprising livelactate utilizing propionic acid producing bacteria, as well as viablelactic acid producing bacteria, its manufacture and use in the treatmentof digestive diseases. US 2014/0341921 discloses species ofhuman-derived bacteria belonging to the Clostridia class that induceaccumulation of regulatory T cells (Treg cells) in the colon andsuppress immune functions, and pharmaceutical compositions containingthese bacteria for use in preventing and treating immune-mediateddiseases such as autoimmune diseases.

SUMMARY OF THE INVENTION

In a clinical trial with infants at risk for atopic disease thedevelopment of faecal microbiota composition and its metabolites wasanalyzed in the first 6 months of life. In infants that had or had notdeveloped eczema when 18 months of age, the differences in intestinalmicrobiota compositions and development thereof earlier in life wasinvestigated. Infants that had an incidence of eczema by the age of 18months showed a decreased acquisition of lactate-utilizing bacteria, inparticular of Eubacterium and Anaerostipes at 26 weeks of age.Furthermore in these infants the amount of faecal lactic acid was low at4 and 12 weeks of age, but increased at 26 weeks of age, when theinfants started receiving complementary feeding (introduction to solidfood). In infants that did not develop eczema this pattern was reversed,in that the amount of lactic acid was decreased at week 26 compared tothe earlier time points.

This study indicates for the first time a link between the functionalityof the microbiota and the expression of allergic phenotypes in infants,in particular eczema. These observations indicate that the type ofmicrobial succession of species and metabolite cross-feeding at specificdevelopmental stages in early life, in particular during weaning whenthe infant is introduced to solid food and a more adult type diet, havelong term health effects on the immune system. The accumulation oflactic acid and/or a relative low number of lactate utilizing bacteriaat 26 weeks of age, can therefore be a predictor for the risk ofdeveloping eczema later in life. Furthermore, these observations aid thedevelopment of optimal nutritional strategies to support gutcolonization of lactic acid utilizing bacteria when the infant has anage of about 6 months or above. Subsequently, of a large range of fibrestested in in vitro fermentation experiments, only a few fibres, namelyinulin, polydextrose and partially hydrolysed guar gum were able tostimulate the growth of the lactate utilizing bacteria Anaerostipes spp.Therefore nutritional supplements or follow on formula or young childrenformula comprising these fibres together with lactate utilizing bacteriacan help to redirect the microbiota and its metabolism to one moresimilar to healthy infants not developing eczema later in life.

DETAILED DESCRIPTION

Thus in one aspect, the invention concerns a nutritional compositioncomprising lactate utilizing bacteria and at least one fibre selectedfrom the group consisting of polyfructose with an average DP of 10 orhigher, polydextrose with an average DP of 10 or higher and partiallyhydrolysed guar gum with an average DP of 10 or higher. Preferably thenutritional composition is for administration to an infant or youngchild.

The invention also concerns a method for preventing allergy or reducingthe risk of allergy by administering a nutritional compositioncomprising lactate utilizing bacteria and at least one fibre selectedfrom the group consisting of polyfructose with an average DP of 10 orhigher, polydextrose with an average DP of 10 or higher and partiallyhydrolysed guar gum with an average DP of 10 or higher, to an infant oryoung child. For certain jurisdictions, this can also be worded as theuse of bacteria and fibre in the preparation of a nutritionalcomposition for administration to an infant or young child forpreventing allergy or reducing the risk of allergy, wherein the bacteriaare lactate utilizing bacteria and the fibre is at least one fibreselected from the group consisting of polyfructose with an average DP of10 or higher, polydextrose with an average DP of 10 or higher andpartially hydrolysed guar gum with an average DP of 10 or higher. Theinvention can also be worded as a nutritional composition, which ispreferably for administration to an infant or young child, comprisinglactate utilizing bacteria and at least one fibre selected from thegroup consisting of polyfructose with an average DP of 10 or higher,polydextrose with an average DP of 10 or higher and partially hydrolysedguar gum with an average DP of 10 or higher for use in preventingallergy or reducing the risk of allergy. Thus in other words, theinvention concerns a nutritional composition comprising lactateutilizing bacteria and at least one fibre selected from the groupconsisting of polyfructose with an average DP of 10 or higher,polydextrose with an average DP of 10 or higher and partially hydrolysedguar gum with an average DP of 10 or higher for use in preventingallergy or reducing the risk of allergy by administering the nutritionalcomposition to an infant or young child.

In a preferred embodiment the nutritional composition according to theinvention, or in the method or use according to the invention, thelactate utilizing bacteria are selected from the group consisting ofAnaerostipes and Eubacterium.

The invention also concerns a method for preventing allergy or reducingthe risk of allergy by administering a nutritional compositioncomprising at least one fibre selected from the group consisting ofpolyfructose with an average DP of 10 or higher, polydextrose with anaverage DP of 10 or higher and partially hydrolysed guar gum with anaverage DP of 10 or higher to an infant or young child. For certainjurisdictions, this can also be worded as the use of fibre in thepreparation of a nutritional composition for administration to an infantor young child for preventing allergy or reducing the risk of allergy,wherein the fibre is at least one selected from the group consisting ofpolyfructose with an average DP of 10 or higher, polydextrose with anaverage DP of 10 or higher and partially hydrolysed guar gum with anaverage DP of 10 or higher. The invention can also be worded as anutritional composition, which is preferably for administration to aninfant or young child, comprising at least one fibre selected from thegroup consisting of polyfructose with an average DP of 10 or higher,polydextrose with an average DP of 10 or higher for use preventingallergy or reducing the risk of allergy. In this embodiment, thenutritional composition preferably further comprises lactate utilizingbacteria. Preferably the lactate utilizing bacteria are selected fromthe group consisting of Anaerostipes and Eubacterium.

In a preferred embodiment of the present invention, the nutritionalcomposition is for administering to or for use in an infant or youngchild that is at enhanced risk of developing allergy.

In a preferred embodiment the infant or young child that is at enhancedrisk of developing allergy is identified by determining theconcentration and/or percentage of lactic acid based on total organicacids in faecal material obtained from the infant or young child

-   -   a. at a first time point when the infant or young child is about        3 to 17 weeks old, and    -   b. at a second time point when the infant or young child is        about 20 to 35 weeks old, and    -   c. subsequently determining whether the concentration and/or        percentage of lactic acid based on total organic acid in the        faecal material has increased or decreased from the first to the        second time point,

wherein an increase in lactic acid indicates an enhanced risk fordeveloping allergy in the infant or young child, and/or by

determining the amount or relative amount of lactate utilizing bacteriain faecal material obtained from the infant or young child

-   -   a. at a first time point when the infant or young child is about        3 to 17 weeks old, and    -   b. at a second time point when the infant or young child is        about 20 to 35 weeks old, and    -   c. subsequently determining whether the amount or relative        amount of lactate utilizing bacteria has increased or decreased        from the first to the second time point,

wherein a minimal increase or a decrease in the amount or in therelative amount of lactate utilizing bacteria indicates an enhanced riskfor developing allergy in the infant or young child.

In one aspect the invention also concerns a method for assessing whetheran infant is at enhanced risk for developing allergy, comprisingdetermining the concentration and/or percentage of lactic acid based ontotal organic acids in faecal material obtained from the infant

-   -   a. at a first time point when the infant is about 3 to 17 weeks        old, and    -   b. at a second time point when the infant is about 20 to 35        weeks old, and    -   c. subsequently determining whether the concentration and/or        percentage of lactic acid based on total organic acid in the        faecal material has increased or decreased from the first to the        second time point,

wherein an increase in lactic acid indicates an enhanced risk fordeveloping allergy in the infant.

Likewise, the invention also concerns a method for assessing whether aninfant is at enhanced risk for developing allergy, comprisingdetermining the amount or relative amount of lactate utilizing bacteriain faecal material obtained from the infant or young child

-   -   a. at a first time point when the infant is about 3 to 17 weeks        old, and    -   b. at a second time point when the infant is about 20 to 35        weeks old, and    -   c. subsequently determining whether the amount or relative        amount of lactate utilizing bacteria has increased or decreased        from the first to the second time point,

wherein a minimal increase or a decrease in the amount or in therelative amount of lactate utilizing bacteria indicates an enhanced riskfor developing allergy in the infant.

In all aspects and embodiments of the present invention, the allergypreferably is eczema.

Microbiota and its Metabolites

It was found that the amount, or relative amount, of lactate utilizingbacteria was decreased in the microbiota of infants at the age of 26weeks, of infants that developed allergy, in particular eczema, whencompared to infants that did not develop allergy. In particular theamount and the relative amount of Anaerostipes and Eubacterium wasdecreased. Anaerostipes is a genus belonging to the family ofLachnospiraeceae, is strictly anaerobic and is a member of the humanintestinal microbiota. It can use lactic acid as a carbon and energysource and produces mainly butyric acid. Known species of Anaerostipesare A. caccae, A. butyraticus A. hadrus and A. rhamnosivorans.Eubacterium is a genus belonging to the family of Eubacteriaceaea, isstrictly anaerobic and some of the species are member of the humanintestinal microbiota. In particular Eubacterium limosum and Eubacteriumhallii are known to utilize lactate usually producing butyrate. In thepresent method for assessing whether an infant is at enhanced risk fordeveloping allergy, preferably the amount or relative amount of lactateutilizing bacteria is the amount or relative amount of lactate utilizingbutyrate producing bacteria. In the present method for assessing whetheran infant is at enhanced risk for developing allergy, preferably theamount of lactate utilizing bacteria is the total of the amount ofAnaerostipes spp and Eubacterium spp. Preferably it is the total of theamount of Anaerostipes caccae, Anaerostipes butyraticus, Anaerostipeshadrus, Anaerostipes rhamnosivorans, Eubacterium limosum and Eubacteriumhallii. In one embodiment in the present method for assessing whether aninfant is at enhanced risk for developing allergy, preferably the amountof lactate utilizing bacteria is the total of the amount of Anaerostipesspp, preferably the total of the amount of Anaerostipes caccae,Anaerostipes butyraticus, Anaerostipes hadrus and Anaerostipesrhamnosivorans. In one embodiment in the present method for assessingwhether an infant is at enhanced risk for developing allergy, preferablythe amount of lactate utilizing bacteria is the total of the amount ofEubacterium spp, preferably the total of the amount of Eubacteriumlimosum and Eubacterium hallii. In the context of the present inventiona minimal increase in the amount of lactate utilizing bacteria means anincrease of less than 0.1% based on total bacteria. A minimal increasein the relative amount of lactate utilizing bacteria means an increaseof less than 0.1%. Other lactate utilizing bacteria are Coprococcuscatus and Veillonella sp. These bacteria mainly produce propionate fromlactate.

It was found that the amount and relative amount, or concentration andpercentage, of D- and L-lactic acid was increased in the faecal materialof microbiota of infants of the age of 26 weeks that developed allergy,in particular eczema, when compared to infants that did not developallergy. At earlier age this was reversed and the amount of lactic acidwas lower at the age of 4 and 12 weeks or younger in infants thatdeveloped allergy. The increased levels of acetate and lactate, early inlife, at 4 and 12 weeks in infants that did not develop allergy, may becrucial for the establishment of the lactate utilizing bacteria around 6months of age. This is indicative that microbial succession of speciesand metabolite cross-feeding at specific developmental stages in earlylife are essential in establishing a gut community and environment thatsupports the immune system. Aberrant temporal dynamics up to 26 weeks ofage in infants developing allergy in the first 18 months of liferegarding microbiota (lactate utilizing bacteria) and its metabolites(levels of lactic acid) was observed. Therefore, remedies to improvethese dynamics towards the dynamics as found in infants not developingallergy, in particular eczema, can be designed. Hence in one embodimentthe invention relates to nutritional compositions comprising lactateutilizing bacteria, preferably lactate utilizing butyrate producingbacteria, preferably Anaerostipes spp and Eubacterium spp, morepreferably at least one selected from Anaerostipes caccae, Anaerostipesbutyraticus, Anaerostipes hadrus, Anaerostipes rhamnosivorans, E. halliiand E. limosum. In one embodiment, preferably the Anaerostipes isAnaerostipes caccae. These species can be isolated from human faeces asknown in the art, or can be ordered from culture collections. The lacticacid utilizing bacteria can be cultured and dried as known in the art.Preferably a nutritional composition comprising lactic acid utilizingbacteria comprises 10² to 10⁹ cfu lactate utilizing bacteria per g dryweight, more preferably 10³ to 10⁶ cfu.

Fibres that Stimulate Growth of Lactic Acid Utilizing Bacteria

In one embodiment the invention relates to a method to increase theamount or relative amount of lactate utilizing bacteria, preferablylactate utilizing butyrate producing bacteria, preferably Anaerostipesand/or Eubacterium, in the intestinal microbiota of an infant or youngchild, said method comprising the administration of at least one fibreselected from the group consisting of polyfructose with an average DP of10 or higher, polydextrose with an average DP of 10 or higher andpartially hydrolysed guar gum with an average DP of 10 or higher to saidinfant or young child.

The term ‘degree of polymerisation’ (DP) as used herein means number ofmonomer units joined together in the poly- or oligomer. The term“soluble” as used herein, when having reference to a polysaccharide,fibre or oligosaccharide, means that the substance is at least 50%soluble according to the method described by Prosky et al., J Assoc OffAnal Chem, 1988, 71:1017-1023. An average degree of polymerizationrefers to the average degree of polymerization based on weight.

Preferably fibre selected from the group consisting of polyfructose withan average DP of 10 or higher, polydextrose with an average DP of 10 orhigher and partially hydrolysed guar gum with an average DP of 10 orhigher is present in a nutritional composition in an amount of 0.2 to 8g based on 100 g dry weight, more preferably 0.5 to 5 gram based on 100g dry weight of the nutritional composition.

According to a particularly preferred embodiment, partially hydrolysedguar gum (PHGG) is used as a fibre to stimulate lactic acid utilizingbacteria or to prevent allergy. Guar gum is a polysaccharide obtainablefrom the endosperm of Cyamopsis tetragonolobus and contains mainly highmolecular weight hydrocolloidal polysaccharide, composed of galactoseand mannose units combined through glycosidic linkages. Specifically,the guar gum preferably consists of linear chains of (1→4)beta-D-mannopyranosyl units with alpha-D-galactopyranosyl units attachedby (1→6) linkages. PHGG is commercially available under the tradenameBenefiber® from Novartis Nutrition Corporation or under the tradename“Sunfiber® AG” from Taiyo Kagaku. Preferably, the hydrolysed gum is inan agglomerated form, which has better solubility. The DP is typicallybetween 10 and 300. Preferably the DP is between 20 and 150, even morepreferably between 25 and 100.

The fibre selected from polyfructose, polydextrose and partiallyhydrolysed guar gum in the present nutritional composition preferablycomprises 0.2 to 8 grams, even more preferably 0.5 to 5 grams partiallyhydrolysed guar gum, based on 100 g dry weight of the nutritionalcomposition.

According to a particularly preferred embodiment, polyfructose is usedas a fibre to stimulate lactic acid utilizing bacteria or to preventallergy. The term polyfructose, or fructopolysaccharide, refers to apolysaccharide carbohydrate comprising a chain of at least 10 β-linkedfructose units with a DP between 10 and 300, preferably between 20 and300. Preferably inulin is used. Inulin is e.g. available under thetradename “Raftilin HP®”, (Orafti). The term “inulin” is used herein torefer to glucose-terminated fructose chains with at least 90% fructoseunits having a DP between 10 and 300. Inulin can be described as GF_(n),wherein G represents a glucosyl unit, F represents a fructosyl unit andn is the number of fructosyl units linked to each other, n being 9 ormore. A small part of the inulin molecules, however, may have noterminal glucose unit, due to hydrolysis during processing. The averageDP of the fructopolysaccharide is preferably at least 10, morepreferably at least 15, more preferably at least 20 or more, up to 300.In inulin the fructose units are linked with a β(2→1) linkage. The fibreselected from polyfructose, polydextrose and partially hydrolysed guargum in the present nutritional composition preferably comprises 0.2 to 8grams, even more preferably 0.5 to 5 grams fructopolysaccharides, basedon 100 g dry weight of the nutritional composition.

According to a particularly preferred embodiment, polydextrose is usedas a fibre to stimulate lactic acid utilizing bacteria or to preventallergy. Polydextrose, or indigestible polydextrin, refer todigestion-resistant (malto)dextrins or digestion-resistant polydextrose,or indigestible starch which have a DP of 10 to 50, preferably between10 and 20. Polydextrose is at least 75% indigestible, preferably atleast 90% indigestible. These polydextrose are preferably produced by acombination of hydrolysis and transglucosidation reactions. In apreferred embodiment, the polydextroses comprise α(1→4), α(1→6)glucosidic bonds and 1→2 and 1→3 linkages. Polydextroses are for exampleavailable under the tradename “Fibersol 2®” from Matsutami Inductries,“Sta-Lite®” Polydextrose from Tate and Lyle, Novelose 330®” fromIngredion, or Litesse® from Danisco. The fibre selected frompolyfructose, polydextrose and partially hydrolysed guar gum in thepresent nutritional composition preferably comprises 0.5 to 8 grams,even more preferably 1 to 5 gram digestion resistant polydextrose, basedon 100 g dry weight of the nutritional composition.

Macronutrients

The present composition preferably contains 5 to 16 en % protein; 35 to60 en % fat; and 25 to 75 en % carbohydrates, preferably 5 to 12.0 en %protein; 39 to 50 en % fat; and 40 to 55 en % digestible carbohydrates.The term “en %” is short for energy percentage and represents therelative amount each constituent contributes to the total caloric valueof the preparation. For example 40 en % digestible carbohydrates equals10 g of carbohydrates per 100 kcal, as 1 g of carbohydrates has 4 kcal.

The composition preferably contains 1.4 to 6 g of a protein source per100 ml. The composition comparably contains 8.5 to 19 g per 100 g dryweight. The protein source may comprise intact protein, hydrolysedproteins, peptides or free amino acids or mixtures thereof. Suitableprotein sources are cow's milk protein, casein, whey and soy protein.The protein content is based on the Kjeldahl percentage, N*6.38.

The composition preferably further contains 2.1 to 6.5 g fat per 100 ml,containing 0.3 to 1.5 g linoleic acid (LA) per 100 ml, at least 50 mgα-linolenic acid (ALA) per 100 ml, in which the ratio of LA/ALA rangesfrom of 5 to 15. Based on dry weight the composition preferably contains12.5 to 30 g fat, 1.8 to 12.0 g LA, and at least 0.30 g ALA per 100 g,in which the ratio of LA/ALA ranges from 5 to 15. The amount ofsaturated fatty acids is preferably between 10 and 58 wt.% of totalfatty acids, the concentration of monounsaturated fatty acids rangesfrom 17 to 60% based on weight of total fatty acids and theconcentration of polyunsaturated fatty acids ranges from 11 to 36% basedon weight of total fatty acids. These amounts and ratios of ALA and LAhave the advantage that a balanced biosynthesis of n-3 and n-6polyunsaturated fatty acids is achieved. Preferably the presentcomposition contains long chain polyunsaturated fatty acids (LC PUFA),such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) andarachidonic acid (AA). Suitable lipid sources are milk fats, canola oil,safflower oil, sunflower oil, olive oil, coconut oil, marine oils, etc.or fractions or mixtures thereof comprising suitable fatty acids.

The present composition preferably contains 6 to 19 g digestiblecarbohydrates per 100 ml, more preferably 6-19 gram lactose.

Nutritional Composition

The present composition is preferably administered in liquid form. Inorder to meet the caloric requirements, the composition preferablycontains 50 to 200 kcal/100 ml, more preferably 60 to 90 kcal/100 ml.Preferably the composition is in a liquid form, with a viscosity below35 cps. When suitable, the composition is in a powdered form, which canbe reconstituted with water to from a liquid. Preferably the nutritionalcomposition according to the present invention or in the method or useaccording to the present invention is a follow on formula or a youngchild formula.

Application

An aberrant temporal dynamics up to 26 weeks of age in infantsdeveloping allergy, in particular eczema, in the first 18 months of liferegarding microbiota, preferably lactate utilizing bacteria, and itsmetabolites, preferably lactic acid, was observed. Therefore, thesedynamics can be used to assess the risk of the infant to improve thesedynamics towards the dynamics as found in infants not developingallergy, in particular eczema, can be designed.

Hence in one embodiment the invention relates to a method of assessingwhether an infant is at enhanced risk for developing allergy, the methodcomprising determining the concentration and/or percentage of lacticacid based on total organic acids in faecal material obtained from saidinfant at about 20 to 35 weeks of age, preferably about 26 weeks of age,and at an earlier time point, when the infant is about 3 to 17 weeks ofage, preferably about 4 to 12 weeks of age, preferably at 4 and/or 12weeks of age. The infant has an increased risk for allergy when theamount, e.g. concentration, and/or percentage of D-lactic acid, L-lacticacid, or the sum of D- and L-lactic acid, is higher at the time point of20 to 35 weeks, preferably about 26 weeks of age, when compared toearlier time points, most preferably at 4 and/or 12 weeks of age. Theinfant has an increased risk for allergy when the amount of L-lacticacid, is above 10 mmol/kg faeces at the time point of 20 to 35 weeks,preferably about 26 weeks of age. The infant has an increased risk forallergy when the relative amount of D-lactic acid, L-lactic acid, or thesum of D- and L-lactic acid is higher at the time point of 20 to 35weeks, preferably about 26 weeks of age, when compared to earlier timepoints, when the infant is about 3 to 17 weeks of age, preferably about4 to 12 weeks of age most preferably at 4 and/or 12 weeks of age. Theamount of lactic acid in the faeces or colon refers to the concentrationof lactic acid in the faeces or colon, e.g. mol/kg or mol/l. Therelative amount of lactic acid relates to the percentage of moles oflactic acid based on total organic acids, being the sum of D- andL-lactic acid, acetic acid, propionic acid, butyric acid, valeric acid,isobutyric acid and isovaleric acid. The infant has an increased riskfor allergy when the relative amount of L-lactic acid, is above 10% atthe time point of 20 to 35 weeks, preferably about 26 weeks of age.

Hence in one embodiment the invention relates to a method of assessingwhether an infant is at enhanced risk for developing allergy, the methodcomprising determining the amount or relative amount of lactateutilizing bacteria, preferably lactate utilizing butyrate producingbacteria, preferably Anaerostipes and/or Eubacterium in faecal materialobtained from said infant at about 20 to 35 weeks of age, preferablyabout 26 weeks of age, and at an earlier time point, when the infant isabout 3 to 17 weeks old, preferably about 4 to 12 weeks of age,preferably at 4 weeks of age. Preferably the total amount ofAnaerostipes caccae, Anaerostipes butyraticus, Anaerostipes hadrus,Anaerostipes rhamnosivorans, E hallii and E. limosum is determined. Theinfant has an increased risk for allergy when the amount of lactateutilizing bacteria is lower, or has minimally increased, at the timepoint of 20 to 35 weeks, preferably about 26 weeks of age, when comparedto earlier time points, when the infant is about 3 to 17 weeks of age,preferably about 4 to 12 weeks of age most preferably at 4 weeks of age.The amount of bacteria relates to the amount of bacteria per ml faeces.Amount of bacteria can also be concentration, or abundance. Relativeamount and relative abundance relates to the percentage of a specificgroup of bacteria based on total bacteria.

In one embodiment the invention relates to a method for preventingallergy or decreasing the risk of allergy by administering lactateutilizing bacteria or fibres that stimulate lactate utilizing bacteriaor both. These fibres and bacteria have been described above in moredetail. Alternatively the invention relates to the administration of oneor more fibres selected from the group consisting of polyfructose withan average DP of 10 or higher, polydextrose with an average DP of 10 orhigher and partially hydrolysed guar gum with an average DP of 10 orhigher to simulate the amount of lactate utilizing bacteria.

Allergy relates to allergic disease, and is a condition caused byhypersensitivity of the immune system to something in the environmentthat usually causes little problem in most people. Allergic diseasesinclude hay fever, food allergies, atopic dermatitis, rhinitis andallergic asthma. In particular allergy was determined based on its skinsymptoms. In a preferred embodiment allergy is eczema. In yet a furtherpreferred embodiment, eczema includes one or more of allergic eczema,allergic dermatitis and allergic eczema dermatitis syndrome (AEDS).

Eczema can be diagnosed based on 2 out of 3 positive scores according tothe modified Hanifin and Rajka criteria (Kunz et al., 1997, Dermatology195(1):10-19)

As the timing of lactate accumulation and lactate utilizing bacteriadepletion is very relevant, the methods or nutritional compositions ofthe present invention are preferably administered to infants that arestarting to being introduced to complementary feeding or solid food. Thediversification of the infant diet around the time of weaning not onlymarks the gradual diversification towards an adult gut microbiota, butalso exposes the infant to an increasing level of antigens from thediet, which requires the immune system to adequately respond to theseharmless substances while safeguarding defense against potentialpathogens. The establishment of lactate utilizing bacteria around 6months of age, e.g. at about 26 weeks, may prove important forestablishing and maintaining homeostasis with our immune system duringthis critical stage of development. Hence, in one embodiment of themethod or use according to the invention, the nutritional composition isadministered to or for administration to an infant and wherein theinfant is being introduced to complementary feeding or solid food.

As the timing of lactate accumulation and lactate utilizing bacteriadepletion is very relevant, the methods or nutritional compositions ofthe present invention are preferably administered to infants that areabout 5 to 18 months of age, more preferably 6 to 12 months of age. Thiscorresponds to the age of weaning.

DESCRIPTION OF THE FIGURE

The FIGURE shows Principal Response Curves representing the changes inbacterial metabolites, i.c. levels of short chain fatty acids (SCFA) andlactic acids, across time and its interaction with developing eczema.

EXAMPLES Example 1 Aberrant Temporal Dynamics in Infants DevelopingEczema in the First 18 Months of Life; Decreased Microbial Conversion ofLactic Acid in Infants Developing Eczema

Faecal samples were taken when infants were 4, 12 and 26 of age. Faecalsamples were collected by the parents into 10 ml stool containers(Greiner Bio-One, Kremsmünster, Austria), immediately frozen (−12° C. to−20° C.) and transported within three months in a cold storage bag withice-packs to the hospital. Upon arrival at the hospital and prior toevaluation at the laboratory, samples were kept and transported atultra-low temperatures (−75° C. to −85° C.).

Frozen stool samples were defrosted on ice and were ten times diluted inPBS buffer (150 mM NaCl, 10 mM Na₂HPO4, 20 mM NaH₂PO₄, pH 7.4) andhomogenized by the addition of 5 to 10 glass beads (3 mm in diameter)and vortexing for 3 min. Glass beads and larger particles were removedby centrifugation at 300×g for 1 min. Several 1 ml portions of thehomogenized suspension were stored at ultra-low temperatures untilfurther processing and analysis.

Faecal suspensions were thawed on ice and centrifuged for 10 minutes at14.000×g. Next, 350 μl supernatant was heated for 10 minutes at 100° C.to inactivate all enzymes and centrifuged again. A portion of thesupernatant was used to quantitatively determine the SCFA acetic,propionic, n-butyric, isobutyric, isovaleric and n-valeric acids by gaschromatography. Another portion of the supernatant was used toenzymatically analyze the levels of lactate using a D-/L-lactic acidassay kit (Megazyme, Wicklow, Ireland).

16S rRNA-gene sequencing was used to characterize the microbiotacomposition of faeces collected at 4 and 26 weeks of age in a set ofvaginally born infants, including breastfed infants (BF, n=30) andinfants receiving the experimental formula (n=51) or control formula(n=57). Diagnosis of eczema at the age of 18 months was based on 2 outof 3 positive scores according the modified Hanifin and Rajka criteria(Kunz et al., 1997, Dermatology 195(1):10-19). Of these 138 infants 52infants had developed eczema when 18 month of age. Faecal samples formicrobial analysis were selected with the following criteria: (I)infants were selected from the breastfed reference group and from thekey group of interest (KGI), which consisted of those infants thatstarted formula before 4 weeks of age, (II) when born vaginally and(III) if stool specimens were available at 4, 12 and 26 weeks of age.

Faecal suspensions were thawed on ice and 200 μl of each suspension wasmixed with 450 μl of extraction buffer (100 mM Tris-HCl, 40 mM EDTA, pH9.0) and 50 μl of 10% sodium dodecyl sulfate. Phenol-chloroformextractions combined with bead-beating were subsequently performed asdescribed by Matsuki, et al., 2004, Appl Environ Microbiol 70:167-173except that DNA was re-suspended in 0.1 ml of TE (10 mM Tris-HCl, 1 mMEDTA, pH 8.0). The V3-V5 regions of the 16S rRNA gene were amplifiedusing the forward primer 357F, and a ‘bifidobacteria-optimised’ reverseprimer 926Rb. The reverse primers included a 12 base-pairerror-correcting Golay barcode. PCR was carried out in quadruplicate aspreviously described in Sim et al., 2012, PLoS One 7: e32543). Replicateamplicons were pooled and purified and three 454 Life Sciences GS FLX(Roche, Branford, Conn., USA) pyrosequencing runs were carried outfollowing the Roche Amplicon Lib-L protocol.

The ‘Quantitative Insights Into Microbial Ecology’ (QIIME) v1.5.0package was used to analyse shotgun processed data (Caporaso et al,2010, Nat Methods 7: 335-336). Sequencing data was first denoised usingAmpliconNoise followed by chimera-removal with Perseus (Quince et al,2011, BMC Bioinformatics 12: 38). Alignment was carried out using theSILVA rRNA database (SSU_REF108) (Pruesse et al, 2007 Nucleic Acids Res35: 7188-7196) for reference and clustered at 97% sequence identity intooperational taxonomic units (OTUs) using the UCLUST algorithm (Edgar,2010 Bioinformatics 26: 2460-2461). Rarefaction was performed andsequences present only once in the dataset (singletons) were removed.

The statistical analyses of the 16S rRNA-gene markers (frequencies ofOTUs) and bacterial metabolite data (levels of SCFAs and lactate) wereperformed using Canoco 5 software (Šmilauer and Lepš, 2014, MultivariateAnalysis of Ecological Data using CANOCO 5, 2nd ed. edn. CambridgeUniversity Press: Cambridge Books Online) and differential abundancetesting using the R-package MetagenomeSeq (Paulson et al., 2013, NatMeth 10:1200-1202). The OTU count data were first aggregated at thegenus level, resulting in a total of 142 genera, normalized by total sumscaling and log-transformed. Genera that were present in less than 10samples were discarded to account for the sparsity of the data. Thisresulted in 58 taxonomic features that were used as input for thestatistical analyses performed. Canoco 5 was used to model the taxonomicdata using constrained ordination methods and Monte Carlo permutationtests (MCPT) to evaluate the explanatory power of sample covariates (TerBraak, 1986, Ecology 67: 1167-1179; Van den Brink and Ter Braak, 1999,Environmental Toxicology and Chemistry 18: 138-148) with significance at0.05. The sample covariates identified were subsequently used in allmodels and comparisons. Benjamini-Hochberg false-discovery rate (FDR)was used to account for multiple comparisons (Benjamini and Hochberg,1995 Journal of the Royal Statistical Society Series B (Methodological)57: 289-300) with significance for adjusted P-values (P-adj) at 0.05.

Constrained ordination methods combined with forward selection ofvariables in Canoco 5 was used to identify the sample covariates thatexplained most of the variation in microbial taxonomic composition(Legendre and Legendre, 2012 Numerical ecology, vol. 24. Elsevier:Amsterdam; Ter Braak, 1986). The approach of forward selectionidentified age (in weeks), feeding group (formula 1, 2 or breastfedreference group), ethnicity (Asian, caucasian or other) and havingsiblings (yes or no) as significantly influencing the bacterialtaxonomic composition of the fecal samples (with Benjamini-Hochbergmethod to control for false-discovery rate with significance at 0.05).The sample covariates identified were subsequently used in all modelsand comparisons, either as explanatory variables or as covariates withsignificance for adjusted P-values (P-adj) at 0.05.

Principal Response Curves (PRC) and the Monte Carlo Permutation test(MCPT with 499 permutaions and significance at 0.05) were used to assessthe effects of developing eczema on the bacterial metabolites (SCFA andlactic acids) over time (Van den Brink and Ter Braak, 1999).Differential abundant bacterial genera were evaluated usingMetagenomeSeq with Benjamini Hochberg false discovery rates withsignificance at 0.05.

Univariate data analysis were performed using GraphPad Prism version6.02 for Windows (GraphPad Software, La Jolla, Calif., USA) applyingMann-Whitney test for two-group comparisons and Kruskall-Wallis withDunn's multiple comparisons test for three or more groups withsignificance at 0.05.

Results

The amount of faecal lactate in time is shown in table 1. At 4 and 12weeks the amount of lactic acid tended to be lower in the infants thatdeveloped eczema, but the difference was not statistically different. At26 weeks however, the amount of D and L-lactic acid was significantlyhigher in infants that developed eczema. A similar result was observedwhen the lacic acid was expressed as % of the total organic acids (thesum of D-lactic acid, L-lactid acid, acetic acid butyric acid, propionicacid, valeric acid, isobutyric acid and isovaleric acid), see table 2.

The effect of developing eczema on the fecal bacterial metabolitecomposition over time was investigated using Principal Response Curves(PRC, Van den Brink and Ter Beek, 1999), while correcting for allsignificant covariates identified. Significant temporal differences onthe first constrained axis produced were observed for the interaction.The differential dynamics were most pronounced from 12 to 26 weeks ofage. Infants developing eczema were characterized by decreased levels ofboth isomers of lactate at 12 weeks, a pattern which was subsequentlyreversed at 26 weeks of age. In the FIGURE the horizontal axisrepresents time and the vertical axis the PRC score values. Infants notdeveloping eczema (NO ECZEMA; dashed line) were used as reference leveland has zero PRC values and so its curve lays over the horizontal axis.The change for infants developing eczema in the first 18 months of life(ECZEMA; solid line -) is shown as a response curve relative to thisreference. The microbial response scores are shown on the separatevertical (one-dimensional) plot. The multiple of the PRC score with theresponse score provides a quantitative interpretation as well as thedirection of the microbial change at the respective time points (4, 12and 26 weeks) in infants developing eczema as compared to those that didnot. Result of the PRC analysis for faecal SCFA and LA are shown for thefirst PRC set, which was significant for the interaction (MCPT: P=0.034,explained variation 84.4%, 499 permutations).

TABLE 1 D-lactic acid and L-lactic acid in mmol per kg of wet weightfaeces Age Lactic acid Statistics NO ECZEMA ECZEMA Summary  4 weeksD-lactic n 83 48 ns acid Mean 2.36 2.78 Median (Q1-Q3) 0.020(0.020-2.92) 0.020 (0.020-4.34) L-lactic n 83 48 ns acid Mean 11.1 7.30Median (Q1-Q3) 0.020 (0.020-9.88) 2.30 (0.020-9.62) 12 weeks D-lactic n81 49 ns acid Mean 3.83 3.35 Median (Q1-Q3) 0.020 (0.020-4.17) 0.020(0.020-4.27) L-lactic n 81 49 ns acid Mean 10.2 6.75 Median (Q1-Q3)0.020 (0.020-14.4) 0.020 (0.020-9.64) 26 weeks D-lactic n 86 52 ** acidMean 2.33 5.39 Median (Q1-Q3) 0.020 (0.020-2.11) 0.020 (0.020-9.28)L-lactic n 86 52 * acid Mean 7.74 14.3 Median (Q1-Q3) 0.020 (0.020-7.88)4.37 (0.020-24.2) n = number of non-missing subjects, Q1 = 25% quartileand Q3 = 75% quartile. Statistical summary is based on Kruskall-Walliswith Dunn's multiple comparisons test comparing infants with eczemaversus infants without at the same age (ns = P > 0.05, * = P ≤ 0.05, **= P ≤ 0.01)

The effect of developing eczema on the fecal bacterial metabolitecomposition over time was investigated using Principal Response Curves(PRC, Van den Brink and Ter Beek, 1999), while correcting for allsignificant covariates identified. Significant temporal differences onthe first constrained axis produced were observed for the interaction.The differential dynamics were most pronounced from 12 to 26 weeks ofage. Infants developing eczema were characterized by decreased levels ofboth isomers of lactate at 12 weeks, a pattern which was subsequentlyreversed at 26 weeks of age. In the FIGURE the horizontal axisrepresents time and the vertical axis the PRC score values. Infants notdeveloping eczema (NO ECZEMA; dashed line - - - ) were used as referencelevel and has zero PRC values and so its curve lays over the horizontalaxis. The change for infants developing eczema in the first 18 months oflife (ECZEMA; solid line -) is shown as a response curve relative tothis reference. The microbial response scores are shown on the separatevertical (one-dimensional) plot. The multiple of the PRC score with theresponse score provides a quantitative interpretation as well as thedirection of the microbial change at the respective time points (4, 12and 26 weeks) in infants developing eczema as compared to those that didnot. Result of the PRC analysis for faecal SCFA and LA are shown for thefirst PRC set, which was significant for the interaction (MCPT: P=0.034,explained variation 84.4%, 499 permutations).

TABLE 2 Relative abundance of D-lactic acid and L-lactic acid as mol %of total organic acids measured Age Lactic acid Statistics NO ECZEMAECZEMA Summary  4 weeks D-lactic n 83 48 ns acid Mean 2.88 4.19 Median(Q1-Q3) 0.080 (0.030-0.58) 0.145 (0.040-8.24) L-lactic n 83 48 ns acidMean 9.93 9.35 Median (Q1-Q3) 0.100 (0.030-14.7) 4.63 (0.0525-12.9) 12weeks D-lactic n 81 49 ns acid Mean 4.26 3.39 Median (Q1-Q3) 0.060(0.030-5.89) 0.060 (0.030-6.51) L-lactic n 81 49 ns acid Mean 10.7 7.41Median (Q1-Q3) 0.200 (0.030-15.7) 0.120 (0.030-12.2) 26 weeks D-lactic n86 52 *** acid Mean 1.96 4.61 Median (Q1-Q3) 0.025 (0.020-2.52) 1.90(0.030-8.58) L-lactic n 86 52 ** acid Mean 7.97 11.8 Median (Q1-Q3)0.030 (0.020-8.03) 5.51 (0.030-18.6) n = number of non-missing subjects,Q1 = first quartile and Q3 = third quartile. Statistical summary isbased on Kruskall-Wallis with Dunn's multiple comparisons test comparinginfants with eczema versus infants without eczema at the same age (ns =P > 0.05, * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001)

So, at 26 weeks of age the level and percentage of lactic acid washigher in the faeces of infants that had developed eczema at 18 monthscompared to infants that had not developed eczema at 18 months, and alsothe level and percentage of faecal lactic acid was increased whencompared to the earlier time points of 4 and 12 weeks. In infants thatdid not develop eczema on the other hand the faecal lactate levels werelower at 26 weeks when compared with the earlier time points.

TABLE 3 Relative abundance (%) of Eubacterium spp Age Statistics NOECZEMA ECZEMA Summary  4 weeks n 82 48 ns Mean 0.00780 0.599 26 weeks n86 52 ns Mean  0.275 (1.15) 0.0996 Difference n (sample pairs) 82 48 *26 weeks − Mean 0.280* (1.17) −0.507 4 weeks n = number of non-missingsubjects; Statistical summary is based on Kruskall-Wallis with Dunn'smultiple comparisons test comparing infants with eczema versus infantswithout eczema at the same age (ns = P > 0.05, * = P ≤ 0.05, ** = P ≤0.01, *** = P ≤ 0.001)

TABLE 4 Relative abundance (%) of Anaerostipes spp Age Statistics NOECZEMA ECZEMA Summary  4 weeks n 82 48 ** Mean 0.00171 0.245 26 weeks n86 52 ns Mean 0.434 0.314 26 weeks − n (sample pairs) 82 48 # 4 weeksMean 0.350 0.0942 n = number of non-missing subjects; Statisticalsummary is based on Kruskall-Wallis with Dunn's multiple comparisonstest comparing infants with eczema versus infants without eczema at thesame age (ns = P > 0.05, # = P ≤ 0.1, ** = P ≤ 0.01)

MetagenomeSeq was used to assess differential abundances of bacterialtaxa over time in infants developing and not developing eczema, as wellas the taxa being differential over time comparing the two groups, whilecorrecting for the covariates identified. The increases over timeobserved for Eubacterium and Anaerostipes spp. were more pronounced forinfants not developing eczema as compared to infants developing eczema,see tables 3 and 4. Both genera are associated with a specialist groupof microbes known to convert lactate together with acetate into mainlybutyrate, hence referred to as lactate-utilizing bacteria (LUB). Thelevels of lactic acid and presence lactic acid utilizing bacteria wereindeed inversely correlated.

The increased levels of acetate and lactate, and decreased amounts ofpropionate and butyrate, early in life, at 4 and 12 weeks, beforestarting weaning, may be crucial for the establishment of LUB likeEubacterium and Anaerostipes spp. around 6 months of age, as observed inthis study for subsequent infants not developing eczema in contrast toinfants developing eczema.

In conclusion, this study indicates for the first time a link betweenthe functionality of the microbiota and the expression of allergicphenotypes in early life. It emphasizes the importance of the early lifemicrobial succession of species and metabolite cross-feeding to developa gut physiology that supports gut development, but also the developmentof normal immune responses towards environmental triggers. Theseobservations could aid the development of optimal nutritional strategiesto support timely gut colonization of keystone species in the graduallydiversifying infant gut.

Example 2 L-Lactate Accumulation in Faeces of Infants that DevelopAllergy

To gain insights into the development of gut microbiota in initiallyhealthy infants who develop allergy in early life and identify plausiblemicrobiota biomarkers of allergic disease, a nested case-control studyof Chinese infants from a large Singaporean birth cohort was performed.The maturation of intestinal microbiota and its metabolism was measuredin 20 pair-matched allergic cases and non-allergic controls during thefirst 6 months of life using 16S rRNA sequencing. The allergic infantswere assessed by means of cumulative incidence of clinical allergysymptoms (eczema episode/allergic rhinitis/food allergy) and SCORADvalues; according to the study eczema workflow up to 12 months of age.At age of 6 months higher levels of faecal L-lactate (12.25 mmol/kg wetweight faeces) were detected in infants that were allergic at 12 monthsof age, compared to the L-lactic acid level in infants that did notdevelop allergy (3.95 mmol/kg wet weight faeces), P<0.05. L-Lactic acidlevels in the group of allergic infants were higher at 6 months whencompared to week 3 and month 3, which is indicative for lactateaccumulation in this group. For non allergic infants it was the otherway around and the amount of faecal L-lactic acid was higher at week 3and month 3.

TABLE 5 L-lactic acid in mmol per kg of wet weight faeces FaecalL-lactic acid NO ECZEMA ECZEMA (mmol/kg ww faeces) Month 3 Month 6 Month3 Month 6 L-lactic acid 9.50 3.95 6.55 12.25

Example 3 Selection of Fibres that Stimulate Lactate Utilizing Bacteria

Fresh faecal samples were collected from four healthy adults, pooled,and divided in smaller portions mixed with glycerol (10%) in ananaerobic cabinet and stored at −80° C. Before the experiment the faecalsamples were defrosted and mixed with the fermentation medium in 1:5 ina falcon tube and a t=0 sample was taken, 6 ml of this suspension wasadded to falcon tube with the substrate of interest and mixed. Thismixed suspension was put in a dialysis tube and the dialysis tube wasadded in a 100 ml bottle filled with 100 ml dialysis medium. Bottleswere closed and incubated at 37° C. A fermentation with no addedcarbohydrates acted as a negative control (blanc), whereas fermentationwith glucose served as a positive control. Fibres and glucose were addedat concentrations of 200 mg per 6 ml of faeces suspension.

19 single fibres and 3 fibre mixtures were tested. Fermentation was at37° C. at 48 h under anaerobic conditions. Starting pH of the buffer was6.3.

Preservative medium: buffered peptone 20.0 g/l, L-cysteine-HCl 0.5 g/l,sodium thioglycollate 0.5 g/l, resazurine tablet 1 per litre, adjustedto pH 6.7±0.1 with 1 M NaOH or HCl. Boiled in microwave. Filled into 30ml serum bottles with 25 ml medium. Sterilised 15 minutes at 121° C.

McBain & MacFarlane medium: buffered peptone water 3.0 g/l, yeastextract 2.5 g/l, mucin (brush borders) 0.8 g/l, tryptone 3.0 g/l,L-cysteine-HCl 0.4 g/l, bile salts 0.05 g/l, K₂HPO₄.3H₂O 2.6 g/l, NaHCO₃0.2 g/l, NaCl 4.5 g/l, MgSO₄.7H₂O 0.5 g/l, CaCl₂ 0.228 g/l, FeSO₄.7H₂O0.005 g/l. Filled into 500 ml Scott bottles with the medium andsterilised 15 minutes at 121° C.

Buffered medium: K₂HPO₄.3H₂O 2.6 g/l, NaHCO₃ 0.2 g/l, NaCl 4.5 g/l,MgSO₄.7H₂O, 0.5 g/l, CaCl₂ 0.228 g/l, FeSO₄.7H₂O 0.005 g/l. Adjusted topH 6.3±0.1 with K₂HPO₄ or NaHCO₃. Filled into 500 ml Scott bottles withthe medium and sterilised 15 minutes at 121° C.

Faecal suspension: the preserved solution of faeces was centrifuged at13,000 rpm for 15 minutes. The supernatant was removed and the faeceswas mixed with the McBain & Mac Farlane medium in a weight ratio of 1:5.

Faecal sample was stored in an RNAlater solution (Ambion, Courtaboef,France) for subsequent bacteria DNA or RNA isolation. The faecal samplewas homogenized and the volume of RNAlater was adjusted to achieve afmal faecal solution of 1:10. 200 μl of this 10-fold dilution was addedto 1 ml of PBS buffer and centrifuged for 5 min at 5000 g. Thesupernatant was discarded and the pellet stored at −80° C. Faecal DNAand RNA was extracted. Determination of bacteria abundance wasdetermined based on the quantification of RNA molecules using primerstargeting mainly 16S rRNA sequences. In order to have a common scale ofcomparison with other bacterial quantification methods, the number ofdetected molecules (RNA or DNA) was given as cell equivalents (CE), forwhich a bacteria culture of a reference strain was used as a standardcurve.

For determination of the target bacteria present in the faecal samples,three serial 10-fold dilutions of the extracted RNA or DNA sample wereapplied to qPCR or RT-qPCR, and CT values in linear range of the assaywere applied to the standard curve generated in the same experiment toobtain the corresponding bacterial count in each nucleic acid sample,and then converted to the count per sample. Different species ofbacteria were analysed amongst which there was one lactate utilizingbacteria, Anaerostipes caccae. Statistical analysis was performed usingthe Mann Whitney method.

Results

At t=0 the amount of Anaerostipes caccae was equivalent to 2*10⁸ cfu/ml.Upon 48 h of fermentation with glucose the amount had doubled to 4*10⁸cfu/ml.

Of the 19 different fibres and 3 fibre mixes tested, a substantialincrease in Anaerostipes caccae compared with the controls T=48 h(glucose) was observed with the following fibres: Orafti® GR (Beneo), aninulin with a DP>10 (5-fold), Orafti® HP (Beneo), a long chain inulinwith an average DP of >23 (5 fold), Benefiber® (Novartis) a partiallyhydrolysed guar gum (PHGG) (4 fold), Sta-Lite® (Tate and Lyle) apolydextrose with DP>10 (5 fold) and Novelose® 330 (national starch) aresistant starch (3 fold). One fibre mixture, rich in inulin, alsostimulated the growth of A. caccae.

No increase or even a decrease compared to the glucose control wasobserved with the other fibres tested, e.g the short chain FOSActilight® (Meiji) and Frutalose® (Orafti both having an average DP<5.Table 6 shows the psotive results for polyfructose with an average DP of10 or higher, polydextrose with an average DP of 10 or higher andpartially hydrolysed guar gum with an average DP of 10 or higher. Forcomparison the result of non-positive short chain fructooligosdacchridesin included.

TABLE 6 Increase of A. caccae upon faecal fermentation of severalfibres. Log increase relative t Fibre Log to t = 48 glucose T = 0 blanc8.3 — T = 48 glucose 8.6 0 T = 48 Orafti ® HP 9.3 0.7 T = 48 Orafti ® GR9.3 0.7 T = 48 Actilight ® 8.7 0.1 T = 48 Novelose ® 330 9.1 0.5 T = 48Sta-Lite ® 9.3 0.7 T = 48 Benefiber ® 9.2 0.6

1. A nutritional composition, comprising lactate utilizing bacteria andat least one fibre selected from the group consisting of polyfructosewith an average degree of polymerisation (DP) of 10 or higher,polydextrose with an average DP of 10 or higher and partially hydrolysedguar gum with an average DP of 10 or higher.
 2. The nutritionalcomposition according to claim 1, wherein the lactate utilizing bacteriaare selected from the group consisting of Anaerostipes and Eubacterium.3. The nutritional composition according to claim 1, wherein the lactateutilizing bacteria are selected from the group consisting ofAnaerostipes caccae, Anaerostipes butyraticus, Anaerostipes hadrus,Anaerostipes rhamnosivorans, E hallii and E. limosum.
 4. The nutritionalcomposition according to claim 1, wherein the lactate utilizing bacteriacomprise Anaerostipes caccae.
 5. The nutritional composition accordingto claim 1, wherein the amount of lactate utilizing bacteria is 10² to10⁹ cfu per g dry weight.
 6. The nutritional composition according toclaim 1, wherein the fibre comprises partially hydrolysed guar gum. 7.The nutritional composition according to claim 6, wherein the partiallyhydrolysed guar gum has a DP between 10 and
 300. 8. The nutritionalcomposition according to claim 1, wherein the fibre comprisespolyfructose.
 9. The nutritional composition according to claim 8,wherein the polyfructose has an average DP between 10 and
 300. 10. Thenutritional composition according to claim 8, wherein the polyfructoseis inulin.
 11. The nutritional composition according to claim 1, whereinthe fibre comprises polydextrose.
 12. The nutritional compositionaccording to claim 8, wherein the polydextrose has a DP of 10 to
 50. 13.The nutritional composition according claim 8, wherein the amount offibre is 0.2 to 8 g per 100 g dry weight.
 14. The nutritionalcomposition according to claim 1, which further comprises protein, fatand carbohydrates, wherein the protein provides 5 to 16% of the totalcalories, the fat provides 35 to 60% of the total calories and thecarbohydrates provide 25 to 75% of the total calories.
 15. Thenutritional composition according to claim 14, wherein the proteinprovides 5 to 12% of the total calories, the fat provides 39 to 50% ofthe total calories and the carbohydrates provide 40 to 55% of the totalcalories.
 16. The nutritional composition according to claim 1, which isa follow on formula or young child formula.
 17. A method of preventingallergy or reducing the risk of allergy in an infant or young childcomprising administering to the infant or young child a nutritionalcomposition.
 18. A method of preventing allergy or reducing the risk ofallergy in an infant or young child, comprising administering to theinfant or young child the nutritional composition according to claim 1.